Mortality and risk factor analysis for Candida blood stream infection: A multicenter study

Candida blood stream infection (candidemia) is severe systemic infection mainly develops after intensive medical cares. The mortality of candidemia is affected by the underlying conditions, causative agents and the initial management. We retrospectively analyzed mortality-related risk factors in cases of candidemia between April 2011 and March 2016 in five regional hospitals in Japan. We conducted bivariate and multivariate analysis of factors including causative Candida species, patients' predisposing conditions, and treatment strategies, such as empirically selected antifungal drug and time to appropriate antifungal treatment, to elucidate their effects on 30-day mortality. The study enrolled 289 cases of candidemia in adults. Overall 30-day mortality was 27.7%. Forty-nine cases (17.0%) were community-acquired. Bivariate analysis found advanced age, high Sequential Organ Failure Assessment (SOFA) score, and prior antibiotics use as risk factors for high mortality; however community-acquired candidemia, C. parapsilosis candidemia, obtaining follow-up blood culture, and empiric treatment with fluconazole were associated with low mortality. Logistic regression revealed age ≥65 years (adjusted odds ratio, 2.13) and sequential organ failure assessment (SOFA) score ≥6 (6.30) as risk factors for 30-day mortality. In contrast, obtaining follow-up blood culture (0.38) and empiric treatment with fluconazole (0.32) were found to be protective factors. The cases with candidemia in associated with advanced age and poor general health conditions should be closely monitored. Obtaining follow-up blood culture contributed to an improved prognosis.     

Isolation of multidrug-resistant Haemophilus influenzae harbouring multiple exogenous genes from a patient diagnosed with acute sinusitis

In paediatric patients, β-lactams and macrolides are widely used to treat acute otitis media and sinusitis, which are often caused by either Streptococcus pneumoniae or Haemophilus influenzae. However, resistant isolates have emerged and are becoming more prevalent. H. influenzae generally acquires antimicrobial resistance by mutation or by expression of β-lactamase. In this study, we isolated H. influenzae from a paediatric patient diagnosed with acute sinusitis. This strain harboured multiple exogenous resistance genes: bla_TEM-1, mef(A) and tet(M). DNA sequencing suggested that both mef(A) and tet(M) had been transferred from S. pneumoniae or another Streptococcus. This typical outpatient had not been exposed to excessive levels of antibiotics and had no underlying diseases, strongly suggesting that this type of resistant isolate could become more prevalent.     

Effect of sodium mercaptoacetic acid on different antimicrobial disks in the sodium mercaptoacetic acid double disk synergy test for detection of IMP-1 metallo-β-lactamase-producing Pseudomonas aeruginosa isolates in Japan

We determined the optimal antimicrobial in the sodium mercaptoacetic acid double disk synergy test (SMA-DDST) for the detection of IMP-1-producing Pseudomonas aeruginosa isolates in Japan and evaluated the performance of the test.Fifty-four P. aeruginosa clinical isolates were tested, including 39 IMP-1 producers and 15 non-metallo-β-lactamase (MBL)-producing carbapenem- and ceftazidime (CAZ)-resistant isolates. The SMA-DDST was performed with CAZ, cefepime (CFPM), imipenem (IPM), meropenem (MEPM), doripenem (DRPM), or biapenem (BIPM)-containing disks. The sensitivity of the SMA-DDST with CAZ, CFPM, IPM, MEPM, DRPM, and BIPM was 39/39 (100%), 36/39 (92%), 18/39 (46%), 8/39 (21%), 19/39 (49%), and 36/39 (92%), respectively. The specificity was 15/15 (100%) for all SMA-DDSTs. This suggests that the isolates may have a resistance mechanism other than MBL production for IPM, MEPM, or DRPM. Since the CAZ resistance mechanism in P. aeruginosa is the same as that of CFPM, but differs from that of carbapenems, we conclude that combining CAZ with BIPM SMA-DDSTs can prevent any failure in the detection of IMP-1-producing P. aeruginosa.     

Diagnosis and treatment of Pneumocystis jirovecii pneumonia in HIV-infected or non-HIV-infected patients—difficulties in diagnosis and adverse effects of trimethoprim-sulfamethoxazole

The clinical characteristics of Pneumocystis jirovecii pneumonia (PCP) in patients with immunodeficiency virus (HIV) infection (HIV-PCP) differ from those in patients without HIV infection (non-HIV-PCP). We analyzed 31 adult HIV-PCP cases and 44 non-HIV-PCP cases between 2008 and 2018. The symptomatic period before the diagnosis was shorter in non-HIV-PCP (5 [3–8] days vs. 29 [14–55] days, P < 0.001) and the overall survival rate was lower in the non-HIV-PCP group (P = 0.022). Serum β-D glucan positivity (72.7% vs. 93.5%, P = 0.034) and Grocott stain positivity for Pneumocystis jirovecii in the bronchoalveolar lavage fluid (4.3% vs. 73.3%, P < 0.001) were significantly lower in the non-HIV-PCP group. This difficulty in laboratory diagnosis possibly resulted in the administration of concurrent antibiotics such as quinolones and macrolides (56.8% vs. 19.4% P = 0.002) in the non-HIV-PCP group. The adverse effects due to trimethoprim-sulfamethoxazole were more frequently observed in HIV-PCP (86.2% vs. 35.3%, P < 0.001). The duration of discontinuation of trimethoprim-sulfamethoxazole was 11 [8–14.5] days in HIV-PCP cases. Co-administration of adjunctive corticosteroid therapy did not mitigate hypersensitivity to trimethoprim-sulfamethoxazole. Our analysis indicated that the characteristics of PCP in patients with or without HIV was quite different. HIV-positive patients with PCP should be monitored closely to avoid adverse effects due to trimethoprim-sulfamethoxazole. Because positivity polymerase chain reaction test for P. jirovecii remained high (91.7%), it is suggested that bronchofiberscopy is warranted for diagnosis of PCP in HIV-negative patients.     

New protocol to detect coronary spastic angina without fixed stenosis

A new combined test, accelerated exercise following mild hyperventilation (HV), was examined to determine whether it is effective at detecting a positive response in patients with pharmacologically-induced coronary vasospasm and near normal coronary arteries. Fifty-eight consecutive patients who underwent both triple non-invasive spasm provocation tests and diagnostic coronary angiography were enrolled. They all had pharmacologically-induced coronary vasospasms and no significant organic stenosis. In these patients, an HV test was performed first, followed by a treadmill exercise test (TET), and finally the new combined test under no medication within 3 days. Of the 58 patients, positive responses were observed in 9 patients to the HV, in 15 to the TET, and in 35 to the newly combined test. The remaining 21 patients had negative responses although the triple sequential tests were perfomed. Thus, the sensitivities of the HV test, TET, and newly combined test were 16% (9/58), 26% (15/58), and 63% (35/56), respectively. Forty-six subjects with near normal coronary arteries and no ACh-provoked spasm served as controls. None of these subjects had positive responses to any of these three tests, and thus their specificity was all 100%. No serious or irreversible complications were seen in this study. We recommend this newly-combined protocol for the induction of coronary artery spasm in patients with vasospastic angina pectoris and without significant stenosis as a diagnostic tool.     

Emergence of vancomycin resistance during therapy against methicillin-resistant Staphylococcus aureus in a burn patient--importance of low-level resistance to vancomycin.

OBJECTIVES: Staphylococcus aureus with low-level resistance to vancomycin (VLSA) which could develop into vancomycin-resistant S. aureus (VRSA) is most important. However, VLSA is difficult to detect by standard laboratory methods. We describe here improved methods to detect VLSA. METHODS: Three methicillin-resistant S. aureus (MRSA) strains, designated Fu6, Fu10, and Fu18, were sequentially isolated from the burn wound site of a patient, during vancomycin therapy. The properties of these strains were compared with those of reference strains Mu3 and Mu50 (previous resistant isolates from other patients). RESULTS: The isolated strains, Fu10 and Fu18, had identical phenotypes and genotypes. The vancomycin resistance of Fu10 was equivalent to that of strain Mu3, whereas Fu18 had much higher vancomycin resistance than Fu10 and Mu3, although reaching the level of Mu50. Fu18 showed similar growth to Mu50 on gradient gels and on Mu3 medium. CONCLUSIONS: Our data indicate that the VLSA developed vancomycin resistance during exposure to vancomycin in vivo. The population analysis of tested VLSA and vancomycin intermediately resistant S. aureus (VISA) indicates that a penem at relatively low concentrations induced a significant increase in the number of vancomycin-resistant subpopulations. Furthermore, we confirmed that gradient gel analysis and Mu3 medium are simple and useful methods for the detection of VLSA judged as VSSA by its conventional MIC alone.     

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D–treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Osteoporosis is a common cause of bone fracture in the elderly, costing billions globally due to fractures leading to long-term disability and subsequent exit from the working population (1, 2). Several treatment options are available for osteoporosis which can be divided into antiresorptives and anabolics ranging from orally dosed small molecules to injectable peptides and biologics (2, 3). Antiresorptive and anabolic agents differ in that antiresorptives inhibit or reduce bone resorption, thereby suppressing bone remodeling, whereas anabolics enhance the rate of bone formation while allowing continued resorption and remodeling of bone tissue. Although both treatments result in increased bone mass, resorption and remodeling are key to the microstructural integrity of bone, and emerging evidence points toward anabolics being more effective in reducing fracture (4–6). Currently, the only anabolics in the clinic are the parathyroid hormone and parathyroid hormone-related peptide mimetics (teriparatide and abaloparatide, respectively) and the sclerostin inhibitor monoclonal antibody (romosozumab). Teriparatide and abaloparatide both cannot be administered over 24 mo in a patient’s lifetime due to the risk for developing osteosarcomas, and romosozumab treatment is recommended for 12 mo due to waning efficacy beyond this duration (7, 8). Therefore, with the ever increasing global median age and associated osteoporosis cases, the development of additional anabolic treatment options are of high importance.Bone resorption and bone formation are regulated through communications between osteoclasts and osteoblasts, respectively (9). Among the paracrine factors involved in this process, axon guidance molecules, such as Semaphorin4D (Sema4D) and Semaphorin3A, mediate the regulation of bone cell differentiation. Sema4D, which is expressed and secreted by mature osteoclasts, inhibits osteoblast differentiation through its receptor PlexinB1 (PlxnB1) expressed on osteoblast surfaces. Binding of Sema4D to PlxnB1 leads to the inhibition of the activation of insulin receptor substrate-1 which is downstream of insulin-like growth factor-1 signaling. In addition, Sema4D controls the spatial distribution of bone-forming osteoblasts through PlxnB1-RhoA signaling (10). Mice with genetic deletion of Sema4D or PlxnB1 as well as mice expressing a dominant-negative form of RhoA in osteoblasts exhibit a high bone mass phenotype due to increased bone formation (11). These findings suggest that inhibiting PlxnB1-Sema4D signaling would lead to a bone anabolic effect through enhancement of osteoblastic differentiation while keeping osteoblasts away from osteoclasts to enable efficient osteoclastic bone resorption.We have previously reported a macrocyclic peptide discovery campaign to identify binding sites on PlxnB1 that inhibit its interaction with Sema4D by means of messenger RNA (mRNA) display in combination with genetic code reprogramming, referred to as the RaPID (Random nonstandard Peptides Integrated Discovery) system (12). We successfully identified a 16-mer thioether-macrocyclic peptide, PB1m6, capable of binding human PlxnB1 (hPlxnB1) with single-digit nanomolar-binding affinity and inhibiting its interaction with Sema4D (13). X-ray structural analysis of cocrystals of PB1m6 and hPlxnB1 revealed that PB1m6 is a negative allosteric modulator of the hPlxnB1-Sema4D interaction by binding a cleft distal to the Sema4D-binding interface of hPlxnB1 while still able to inhibit the hPlxnB1-Sema4D interaction. However, PB1m6 was shown to be selective toward hPlxnB1 over mouse PlxnB1 (mPlxnB1) displaying no detectable affinity (dissociation constant (K_D) over 1 µM) regardless of having 88% sequence identity in the N-terminal sema domains of hPlxnB1 and mPlxnB1. Modeling efforts based on the three-dimensional (3D) structure to rationally increase the species cross-reactivity were unsuccessful in our hands. This high selectivity is often observed with RaPID-derived macrocyclic peptides and is generally considered beneficial (13–18). However, in this instance, the high selectivity of PB1m6 hinders its ability to validate the inhibitory mechanism in mouse models. In this study, we used a fragmented saturation mutagenesis approach to create an mRNA library of PB1m6 analogs to be utilized in a RaPID selection campaign against mouse PlxnB1. After five iterative rounds of selection, we discovered a PB1m6 analog, referred to as PB1m6A9, exhibiting enhanced cross-reactivity with 44 nM K_D against mouse PlxnB1 and which, remarkably, also showed 10-fold stronger binding affinity against human PlxnB1 (0.28 nM K_D). To further improve apparent affinity and inhibitory activity, a homodimer of PB1m6A9 was chemically synthesized (PB1d6A9), and it was shown to exhibit potent mPlxnB1-Sema4D inhibitory activity in mouse primary osteoblasts as well as enhanced osteogenesis even when compared to cells not treated with Sema4D. Moreover, once-weekly intravenous (i.v.) administration of palmitoylated PB1d6A9 (PB1d6A9-Pal) in a mouse model of postmenopausal osteoporosis showed significant enhancement of bone formation compared to both vehicle and sham-operated (Sham) control mice. This work presents the facile development of a novel bone anabolic modality which shows promise as an addition to the current repertoire of anabolic agents used to address osteoporosis.     

Comparative sequencing of human and chimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence

Despite their high degree of genomic similarity, reminiscent of their relatively recent separation from each other ( approximately 6 million years ago), the molecular basis of traits unique to humans vs. their closest relative, the chimpanzee, is largely unknown. This report describes a large-scale single-contig comparison between human and chimpanzee genomes via the sequence analysis of almost one-half of the immunologically critical MHC. This 1,750,601-bp stretch of DNA, which encompasses the entire class I along with the telomeric part of the MHC class III regions, corresponds to an orthologous 1,870,955 bp of the human HLA region. Sequence analysis confirms the existence of a high degree of sequence similarity between the two species. However, and importantly, this 98.6% sequence identity drops to only 86.7% taking into account the multiple insertions/deletions (indels) dispersed throughout the region. This is functionally exemplified by a large deletion of 95 kb between the virtual locations of human MICA and MICB genes, which results in a single hybrid chimpanzee MIC gene, in a segment of the MHC genetically linked to species-specific handling of several viral infections (HIV/SIV, hepatitis B and C) as well as susceptibility to various autoimmune diseases. Finally, if generalized, these data suggest that evolution may have used the mechanistically more drastic indels instead of the more subtle single-nucleotide substitutions for shaping the recently emerged primate species.